Carcinogenesis is a multistage event affected by a variety of genetic and epigenetic factors and is typified by the outbreak of uncontrolled cell growth originated from different tissues. A universal goal for anticancer research lies in the development of a clinical treatment that is highly effective in curtailment of tumor growth, non-toxic to the host, and is affordable for most patients. Drugs that inhibit targets that are unique to dividing cells, particularly cells dividing in an uncontrolled manner, are an ideal paradigm for chemotherapeutic agents, the greater the specificity to cells that are dividing in an uncontrolled manner the lower the risk of attendant side effects.
Under normal conditions cells in our bodies are involved in a balanced system of programmed growth, division, rest, and death. The regulation of these cellular pathways is essential in order to maintain tissue viability and bodily health. The transition of a healthy cell to a precancerous or cancerous cell is initiated by the disruption of these regulatory pathways. Cancer cells then redirect the cellular systems to allow for uncontrolled cell growth, replication, and/or resistance to programmed cell death (apoptosis).
Caspases are one means of inducing apoptosis. Apoptosis is regulated by the inhibitor of apoptosis protein (IAP) family of proteins, through their inhibition of caspase-induced cell death. One of the IAP family members, survivin, is overexpressed in pre-cancerous and cancerous cells, and rarely found in healthy adult cells. By their high survivin expression, tumor cells are prevented from entering the caspase-induced cell death pathway that would lead to their destruction. Survivin is one of the targets currently being tested for anticancer therapy.
Another mechanism by which tumor cells grow uncontrollably is by deregulating their cell cycle process. Cdc2 (cyclin-dependent kinase-1) is one of several kinase proteins controlling cell division and is frequently de-regulated in cancer cells. Cdc2 is also involved in the activation of survivin. In addition, growing tumors require a constant supply of essential nutrients and oxygen. Cancer cells achieve their nutrient needs by secreting Vascular Endothelial Growth Factor (VEGF) to induce new blood vessel formation within the tumor mass.
Proteins such as those mentioned above are involved in the regulation of the signaling pathways that control cell growth, division and death. Some of these proteins are significantly altered in cancer cells. The process of converting the gene sequence on DNA to an RNA message (a process called transcription) that can then be converted to protein (a process called translation) is essential to the regulation of protein production.
Cells pass through many checkpoints as they proceed through the cell cycle. Certain criteria must be met in order to pass each of these checkpoints. In the G2/M transition, the most essential regulator is the cyclin-dependent kinase CDC2. This kinase binds tightly to the regulatory protein cyclin B, and this complex, also called the maturation promoting factor (MPF), is responsible for stimulating a myriad of events that lead to the cell's entry into early prophase (1). Not surprisingly, the loss or deactivation of either component of the MPF will block cellular progression out of G2.
The expression and activity of the MPF is regulated at different levels. Cyclin B protein levels slowly rise through the G1 and S phases of the cell cycle, peak during the G2 to M phase transition, and drop sharply during mitosis (2). The CDC2 protein, on the other hand, is always present during the cell cycle, although levels rise slightly in the last stages of the G2 phase (3). The activity of the protein is dependent on the association with the appropriate cyclin, as well as on the dephosphorylation of its inhibitory sites by the phosphatase CDC25C (4, 5). It has been shown that the failure of this dephosphorylation initiates G2 arrest in response to DNA damage by radiation or chemical action. Recent evidence also suggests that any remaining active CDC2 may be transported outside the nucleus following DNA damage (6).
Survivin is an inhibitor of apoptosis that is abundantly expressed in many human cancers (7), but not in normal adult human tissue, and is considered a possible modulator of the terminal effector phase of cell death/survival. (8). Survivin is expressed in G2-M in a cell cycle-dependent manner, binding directly to mitotic spindle microtubules. It appears that survivin phosphorylation on Thr34 may be required to maintain cell viability at cell division (9), and expression of a phosphorylation-defective survivin mutant has been shown to trigger apoptosis in several human melanoma cell lines (10), Phosphorylated survivin acts on the caspase pathway to suppress the formation of caspase-3 and caspase-9, thereby inhibiting apoptosis. (11) Although compounds that reduce the expression of survivin will be expected to increase the rate of apoptosis and cell death, CDC-2 has been shown to be necessary for survivin phosphorylation (9). In addition, the activation of caspases is a time-dependent event as it occurs slowly, quite often inefficiently.
A number of naturally occurring derivatives of the plant lignan nordihydroguaiaretic acid (NDGA) have been shown to block viral replication through the inhibition of viral transcription. NDGA is extracted from the resin of the leaves of Larrea tridentata, a desert bush indigenous to the southwestern US and Mexico. Derivatives of NDGA can inhibit the production of HIV (12, 13), HSV (14), and HPV transcripts (15) by the deactivation of their Sp1-dependent promoters. Isolation and purification of plant lignans, however, is labor intensive and costly. In anticipation of the possible clinical use of plant lignans in controlling Sp1-regulated viral and tumor growth in humans, nine different methylated NDGA activities were synthesized chemically using unmethylated NDGA as the parent substrate in large quantities with low cost (12).
Nordihydroguaiaretic acid (M4N, EM1421, Terameprocol), is the synthetic tetra-methylated derivative of nordihydroguaiaretic acid (tetra-O-methyl nordihydroguaiaretic acid, abbreviated as M4N), The chemical structure of M4N was designed to make it pharmacologically distinct from NDGA. M4N has been shown to possess antiviral (12, 14) and anti-cancer (16) activities in cultured cells, in mouse models (16, 17), and in human xenografts in nude mice (18). M4N causes cell cycle arrest at the G2 phase of the cell cycle probably by suppressing Sp-1 regulated cdk expression (16, 19). M4N has been in Phase I clinical trials in patients by intravenous infusion (CLINICAL TRIALS.GOV, A service of U.S. NIH).
Nordihydroguaiaretic acid (NDGA) derivatives such as M4N suppress Sp1 regulated transcription of viral genes, by deactivation of Sp1-dependent promoters. SP1 also affects expression of many growth-related genes. Cdc2 (also referred to as CDK1) and cyclin B interact to allow cells to move from the G2 phase of cell division to mitosis. M4N blocks the transcription of Cdc2, and thus blocks cell division. The Sp1 protein on promoter of CDC2 chromatin is replaced following M4N treatment in vivo.
M4N is able to induce cell cycle arrest in mammalian cell lines; M4N is a transcription inhibitor. It selectively reduces transcription of growth related genes that have promoters controlled by the Sp1 factor, such as cdc2, survivin and VEGP. By blocking production of cdc2, and VEGF, M4N inhibits tumor growth and starves tumors by restricting growth of their blood supply.
M4N has been shown to arrest growth of a variety of human cells in vitro, the majority of which are part of the NCI panel of 60 cancer cell lines, including solid tumor cell lines (bladder, breast, colorectal, liver, lung, ovarian, pancreatic, prostate and cervical carcinomas), and erythroleukemia cells. In vivo, M4N also decreases tumor cell growth and exhibits antitumor activity in a large number of tumor xenograft models, including human bladder, breast, colorectal liver, ovarian, pancreatic, prostate and cervical carcinomas, and erythroleukemia, without apparent toxicity. M4N has a broad spectrum of activity in anti-cancer therapy, having affects on Cdc2, HIF-1α, MDR1, VEGF and survivin. For example, M4N induces apoptosis and reduces cdc2 protein levels in human oral cancers. M4N also appears to reduce survivin levels in these cancers. Administered systemically, M4N was also shown to inhibit xenografted human tumors MCF-7, Hep3b, LNCaP, HT-29, and K562. Although none of the xenograft tumors were fully eradicated.
M4N does not appear toxic to animals. For example, M4N retention in mouse organs following oral administration has been studied after short term and long term feeding, the results showed essentially no toxic effects even at concentrations high as 906 μg/g of tissue. On daily (1 mg/day) IV injection of M4N for days, M4N accumulated in blood and tumors to levels above 1 mM in nude mice carrying human tumor xenografts.
By use of gene array studies with 9600 expressed genes. Applicants previously found products of most Sp1 regulated genes remained at similar levels, and not affected by the drug treatment of cervical cancer cells C3 in culture.
M4N has some favorable therapeutic qualities, in that it exhibits efficacy against several tumors, by inhibiting cell growth. However, in human clinical trials, treatment with M4N does not generally eradicate disease, and upon cessation of treatment with M4N tumors are capable of growing back. Accordingly, there is a need to identify ways to boost the efficacy of the M4N when the type of cancer is aggressive, metastatic or when M4N as a single drug in low concentration is not enough to induce rapid apoptosis of such type of cancers.
This application claims priority to U.S. Provisional Patent Application 60/010,371, filed Jan. 8, 2008, and U.S. Provisional Patent Application 61/191,827, filed Sep. 12, 2008, each of which is incorporated by reference in its entirety.